THROMBOSIS RESEARCH 71; 139-148,1993
0049-3848/93
$6.00 + .OO Printed in the USA.
Copyright (c) 1993 Pergamon Press Ltd. All rights reserved.
SUPPRESSIVE
EFFECT OF HUMAN BLOOD COAGULATION
FACTOR XIII
ON THE VASCULAR
PERMEABILITY
INDUCED BY ANTI-GUINEA
PIG
ENDOTHELIAL
CELL ANTISERUM
IN GUINEA PIGS
Keizo Hirahara, Kazuhiko Shinbo, Mikiko Takahashi
and Tetsuro Matsuishi
Pharma Research Laboratories, Hoechst Japan Limited, l-3-2,
Minamidai, Kawagoe-shi, Saitama, 350 Japan
(Received 12.11 .1992; accepted in revised form 20.4.1993 by Editor A. Takada)
Abstract
We investigated the effect of blood coagulation factor
XIII(FXII1) on enhanced permeability induced by anti-
endothelial cell antiserum, that was produced by the
immunization
of guinea pig endothelial
cells with
adjuvant into rabbits repeatedly.
We have found that
this
antiserum
reacts
to
human
and
guinea
pig
endothelial cells but not guinea pig fibroblast cells.
The permeability was enhanced by intradermal injection
of 400-fold dilution of this antiserum into dorsal skin
of guinea
pigs.
The mixture
of equal volume
of
antiserum
and FXIII was intradermally
injected into
dorsal skin of guinea pig after Evans blue injection,
and 15 minutes later the quantity of Evans blue at the
each injection site was determined.
We recognized the
suppressive effect of FXIII on the dye leakage. We also
studied the suppressive effect on swelling induced by
the antiserum.
After the subcutaneous injection of the
mixture of antiserum and FXIII into the back of guinea
pigs,
we measured
the thickness
of skins
at the
injection site after day 1, 2 and 3. As a result, FXIII
significantly suppressed the swelling.
We found that
FXIII suppresses the acute and subacute permeability
enhancement.
These results suggest that FXIII plays an
important role on an inflammatory site and that it may
exert as an anti-inflammatory protein.
Blood
coagulation
factor XIII (FXIII),
Key words: Factor XIII, anti-endothelial
permeability, anti-inflammatory protein,
139
the
last
enzyme in the
cell antiserum, vascular
Schonlein Henoch purpura
140
PERMEABILITY
INHIBITION BY FXIII
Vol. 71, No. 2
blood coagulation cascade, is a transamidase that catalyzes the
formation of y - gultamyl - E: - lysyl peptide crosslinks between
polypeptide chains in adjacent fibrin monomers and other plasma
proteins(l,2,3).
Crosslinks
of each fibrin molecule caused a
marked increase in the rigidity of the clot network(4).
On the
other hands, the crosslinks between fibrin and cellular matrix
protein such as fibronectin may exert to connect fibrin molecules
with the injury sites(5).
It is well known that clots play an
important role in the prevention of further tissue damage and in
subsequent wound healing(6).
Schijnlein Henoch purpura (SHP) is
characterized
by hemorrhagic
skin lesions, abdominal
symptoms
including
gastro-intestinal
bleeding,
renal involvement
with
proteinuria and hematuria and swelling of joints(7).
The symptoms
are ascribed
to generalized
inflammation
of arterioles
and
capillaries.
That is, the local changes of the coagulation and
fibrinolytic
system due to immunoreaction
were induced in the
affected vessels.
In 1977, Henriksson and colleagues described a
lowering
of FXIII
activity
during
the acute phase
of this
disease(8).
The mechanism of the decrease of FXIII activity in
the acute phase of SHP has not yet been clarified.
Destruction of
FXIII molecules by protease derived from leukocytes which migrated
into the inflammation
sites has been proposed(9).
In this
connection,
Kamitsuji
and Fukui
et al. reported
that
the
administration
of FXIII
concentrate
may contribute
to the
improvement
of
gastro-intestinal
complications
of
SHP
patients(l0).
Recently FXIII concentrate (Fibrogammin P) is used
for the treatment of SHP patientstll).
According to Matsuoka (121,
Bowie et a1.(13) and Ito et a1.(14), this vasculitis of SHP is
regarded
as the immunovascular
disease that antibody-antigen
complexes on the vascular capillary endothelial cells enhances the
vascular permeability. Consequently non-thrombocytopenic
purpura
caused by the injection of anti-endothelial
cell antiserum(l5).
In the present study, we investigated whether or-not human FXIII
suppresses the enhancement of permeability and swelling induced by
anti-endothelial cell antiserum in guinea pigs.
MATERIALS AND METHODS
Materials
Materials were purchased from the following suppliers: Dulbecco
phosphate buffer, Dulbecco MEM, FCS(Gibco, USA), ECGS(Calbiochem,
USA), Freund's adjuvant(Difco, USA), FITC conjugated anti-rabbit
IgG(Cappe1,
USA), Evans blue, potassium
hydroxide(KOH,
Kanto
Kagaku, Japan), phosphoric acid(Wako Pure Chemical, Japan), Guinea
pig complement(Kyokuto,
Japan), and Human FXIII(Fibrogammin
P,
Behringwerke, FRG).
Vol. 71, No. 2
PERMEABILITY INHIBITION BY FXIII
141
Preparation of anti-guinea pig endothelial cell antiserum
Guinea pig endothelial cells were isolated from the main artery
and vena cava(l6), then cells were inoculated into tissue culture
dishes and incubated for several days with Dulbecco MEM containing
15% FCS and 37.5 pg/ml ECGS till reaching confluency. Confluent
monolayer was harvested by a cell scraper.
The cells were rinsed
twice with Dulbecco phosphate buffered saline(pH 7.2). These cells
were used as an antigen for the production of anti-endothelial
cell antiserum.
The antiserum was obtained from rabbits immunized
with emulsion
of Freund's
complete
adjuvant
with guinea pig
endothelial
cells,
and boosted
with
emulsion
of Freund's
incomplete
adjuvant.
After
several times of boosting,
the
antibody titer was measured with guinea pig endothelial cells by
the methods
of cytolysis
and
indirect
immunofluorescence
microscopy using FITC conjugated anti-rabbit IgG(17).
Measurement of antibody titer of anti-endothelial cell antiserum
Confluent monolayer of guinea pig endothelial cells in a 96-well
plate was incubated with 50 ~1 of variously diluted antiserum in
Dulbecco MEM-15% FCS for 30 min.
The medium was then replaced to
50 11 of 5% guinea pig complement in Dulbecco MEM-15% FCS and the
cells were further incubated for 30 min. After addition of 10 ~1
of trypan blue solution, the cell layers were photographed
to
evaluate the extent of cell lysis.
Indirect immunofluorescence
microscopy was done as follows. The antiserum was serially diluted
two times.
The diluted antiserum was then incubated with the main
artery at room temperature for 1 hour and rinsed 3 times with
Dulbecco phosphate buffer.
After washing, lOOO-fold dilution of
FITC conjugated
anti-rabbit
IgG was added to the sections,
incubated for 30 minutes at room temperature, and washed 3 times
with Dulbecco phosphate buffer.
All sections were observed by a
Nikon microscope equipped with a mercury lamp.
The titer was
taken as a highest dilution which gave a fluorescent staining just
above the background staining of normal serum controls.
Duration of activity of permeability enhancement
Measurement of permeability was studied according to Yamamoto et
al. (18) .
A 100 ~1 portion
of 50-fold diluted antiserum
was
intradermally
injected into the back of a guinea pig before
intravenous
injection of 0.5 ml of 1 % Evans blue.
After 15
minutes of the Evans blue injection, the back skins were harvested
and the blue lesions were observed.
Suppressive effect of FXIII on the permeability enhancement
A 100 ~1 portion of either each diluted antiserum or the mixture
of equal
volume
of FXIII
and the
diluted
antiserum
was
142
PERMEABILITY INHIBITION BY FXIII
Vol. 71, No. 2
intradermally injected into the dorsal skin of guinea pigs after
intravenous injection of Evans blue. After 15 minutes, skins were
harvested and blue lesions in the skins were observed.
Extraction of Evans blue from guinea pig skins
Evans blue was extracted from skins, soaked with 1 ml of 1 M KOH
solution, and incubated at 37'C overnight.
After the incubation,
3 ml of 0.6 N phosphoric acid and 3 ml of FRIGEN(Behringwerke,
FRG), a defatting agent, was added to each tube and mixed for 30
sec. with a Vortex mixer.
Each tube was centrifuged at 3000 rpm
for 15 minutes, and the absorbance of the supernatant was measured
at 620 nm(19).
Suppressive effect of FXIII on the swelling
One milliliter of equal volume mixture of FXIII and the intact
antiserum was subcutaneously
injected into the dorsum of guinea
pigs.
After days 1, 2 and 3, the skins were harvested and the
thickness was measured with a slide caliper at injection sites as
a marker of swelling.
The swelling was shown by the difference of
the thicknesses between a injection and a non-injection site.
RESULTS
Characterization
of polyclonal anti-guinea pig endothelial cell
antiserum
The antibody titer was determined with guinea pig endothelial
cells by the methods of cytolysis and indirect immunofluoresence
microscopy using FITC conjugated anti-rabbit IgG.
As a result,
the 50% cytolysis
was observed
by the 60-fold
dilution
of
antiserum, and the fluorescence was observed by 400-fold dilution.
The antiserum exhibited the reactivity with not only guinea pig
but also human endothelial cells.
However it did not react with
guinea pig fibroblasts.
When the cryosection of the main artery
of a guinea pig was used for the indirect immunofluorescence test,
the fluorescence
was observed on the inner membrane which was
seemed to be endothelial
cell.
It was also found that the
antiserum reacted with the extracellular matrix proteins produced
by endothelial cells(data not shown).
Enhanced permeability
First, we studied whether this antiserum induced the permeability
in guinea pigs.
The variation of permeability after intradermal
injection
is shown in Fig. 1.
The permeability
reached the
maximum
within
5 minutes.
This activity
for enhancing
the
permeability
almost disappeared
within
30 minutes
after the
injection.
This permeability enhancing phenomenon was classified
as a short lasting reaction.
We next investigated
the dose
response of this antiserum.
As shown in Fig. 2, the activity of
Vol. 71, No. 2
PERMEABILITY
INHIBITION
BY FXIII
143
Time course of permeability enhancement induced by anti
-endothelial
cell antiserum.
Antiserum was injected
into a guinea pig at varying times before intravenous
dye injection.
Time 0 means an intradermal injection
immediately
after intravenous
dye injection.
(A) :
antiserum,
60
min,
(B) :
antiserum,
30
min,
(C) :
antiserum,
0 min,
(D) :
saline,
30
min,
(E) :
rabbit
serum, 60
min, (F): rabbit serum, 30
min, (G): rabbit
serum, 0 min, (H): saline, 0 min
FIG. 1.
enhancing the permeability is recognized by 400-fold dilution of
antiserum.
The effect of FXIII was examined on the vascular
permeability induced by the antiserum.
In this experiment, the
mixture of antiserum was injected with various concentration of
FXIII.
As shown in Fig. 3, FXIII shows the suppressive effect on
the dye leakage in a dose dependent fashion. We obtained a result
that both 200-fold and 400-fold diluted antiserum exhibit the same
tendency.
Thus the effect of FXIII was examined in 10 guinea pigs
and the dye leakage was measured in extravascular space. As shown
in Fig. 4, FXIII exhibited the suppressive
effect in a dose
dependent manner.
Suppressive effect of FXIII on the swelling
When the antiserum was subcutaneously injected into a dorsal skin
of guinea pig, edema, in addition to hemorrhage was observed at
injection site(20).
Thus we examined the suppressive effect of
FXIII on the swelling.
On injecting the mixture of FXIII and
antiserum, the edema was significantly suppressed by FXIII on day
1 and 2(Fig. 5).
This result indicates that FXIII suppresses the
permeability in the acute and the subacute phase as well.
144
PERMEABILITY
INHIBITION BY FXIII
Vol. 71, No. 2
FIG. 2.
l/800
l/1600
NRS l/50
buffer cont.
Dose response of anti-endothelial
cell antiserum in a
guinea pig. Each sample was injected immediately after a
intravenous dye leakage.
NRS: Normal rabbit serum
DISCUSSION
For more than 20 years after its detection of FXIII, many authors
have reported that a clotting factor, FXIII, influenced a lot of
other systems and thus it was often termed a connective tissue
factors
(21).
The fibrin stabilizing effect is an example of
general properties of this factor which crosslinks proteins with
suitably configurated E - lysyl- and y - glutamyl - residues. Many
kinds of proteins
are listed as substrates
for FXIII,
e.g.
fibrin(l),
collagen(221,
fibronectin(51,
actin(23)
and factor
V(24).
In this context,
the binding
of biogenic
amines to
proteins by FXIII may also participate in the elimination of toxic
substances like histamine.
FXIII concentrate has been recently
used not only for the promotion of the wound healing but also for
the treatment of Shijnlein Henoch Purpura(SHP) (6,101. The clinical
effects of FXIII on SHP are probably due to the stabilization of
microvasculature
leading
to a reduction
of the leakage
at
inflammatory
sites.
Pilger et al(25) has reported that FXIII
shows the suppressive/sealing
effect in a screlodermia patient.
However none of these reports showed the sealing/suppressive
effect on the permeability
by FXIII in animal studies.
This
vasculitis of SHP is regarded as the immunovascular disease that
Vol. 71, No. 2
PERMEABILITY
INHIBITION
BY FXIII
145
the vascular permeability
is enhanced by the formation of the
antigen-antibody
complex not with standing ambiguity of trigger
which may include
drugs,
foods,
insect
bites
or bacterial
infections(ll,12,13,14).
Thus we tried to demonstrate
the
suppressive effect of FXIII on permeability enhancement induced by
anti-endothelial cell antiserum.
As shown in Figs. 1 and 2, anti-
endothelial
cell
antiserum
induces
the
enhancement
of
permeability.
This phenomenon can be caused by factors such as
complement fragments and histamine etc. which are produced by the
activation
of complement
system after complex
formation
of
antiserum with endothelial cells(ll,l2,13,14) .
As this phenomenon
shows the dose dependent manner by antiserum, condition of SHP
patients may be influenced seriously depending on the extent of
the antibody generation.
SHP patients show the increase of plasma
level of IgA and the imbalance
of serum
IgG subclass
and
IgM(13,14,26) .
As shown in Figs. 3,
4 and 5, FXIII suppresses the
vascular permeability
in acute phase and the edema in subacute
phase.
These results are supported by some clinical studies.
Kamitsuji et al.(lO) and Fukui et al.(ll) have reported that FXIII
shows the suppressive effect on the swelling of joints of SHP
patients.
l/ZOO
diluted
/
ted Ab
FIG. 3.
Suppressive
effect
of FXIII
on the
permeability
enhancement induced by anti-endothelial cell antiserum.
FXIII
was used with the final
concentration
at a
injection site of (A), 3.0 U; (B), 1.5 U; (C), 0.75 U;
(D), 0.38 U; (E), medium control.
The mixture of FXIII
and either
200-
or 400-
fold diluted
antiserum
was
injected immediately after the intravenous injection of
dye.
146
PERMEABILITY
INHIBITION BY FXIII
Vol. 71, No. 2
100 -
80 -
% 6o
40 -
20 -
0'
a-GPEC
+1.5U
nFXlll +3.OU
nFXlll
Sample
FIG. 4
Suppressive effect of FXIII on the permeability induced
by
anti-guinea
pig
endothelial
cell
antiserum.
Extraction
of Evans blue at the injection
site was
according to the materials and methods.
n=lO, *: p<o.os,
**: p<O.Ol
In this experiment, we used the 300-fold
diluted anti-endothelial cell antiserum as a permeability
inducer.
FXIII was mixed with antiserum,
then the
mixture was injected intradermally.
2.0
-l
1.8-
0.8 1
0.6
-I
??
T/f
0.0 I
I
I
I
I
0
1
2
3
4
day
FIG. 5.
a-GPEC
+FXIII
Effect of FXIII on the swelling induced by anti-guinea
pig endothelial cell antiserum.
Open circle (0) denotes
the antiserum
alone.
Closed circle
(0) denotes the
FXIII plus antiserum.
n=5, *: pco.05, **: p,O.Ol
Vol. 71, No. 2
PERMEABILITY INHIBITION BY FXIII
147
Pilger et a1.(25) reported that FXIII also shows the suppressive
effect on vascular permeability in sclerodermia patients. These
results suggest that FXIII may crosslink cellular matrices to
prevent the opening of the space between cells(27) and that it may
crosslink the enhancing factors for the permeability(21).
We have
succeeded in demonstrating
the suppressive effect of FXIII on
vascular permeability in an animal study.
This study indicates
that FXIII may play a crucial role in an inflammatory
site.
Consequently it seems that FXIII therapies are necessary for the
treatment of some inflammatory diseases (28,29).
REFERENCES
1. ROBBINS, K.C. A study of the comparison
of fibrinogen to
fibrin.
AM.
J. Physiol., 142, 581-588, 1944
2. KESKI-OJA, J., MOSHER, D.F. and VAHERI, A. Crosslinking of a
major surface-associated glycoprotein (fibronectin) catalyzed by
blood coagulation factor XIII. Cell, 9, 29-35, 1976
3. ICHINOSE, A., TAMAKI, T. and AOKI, N. FXIII mediated cross-
linking of NH2-terminal peptide of alpha-2 plasmin inhibitor to
fibrin. FEBS-letter, 153, 369-371, 1983
4. SHEN, L. and LORAND, L. Contribution of fibrin stabilization
to clot strength. J. Clin. Invest., 71, 1336-1341,1983
5. OKADA, M., BLOMBACK,
B., DER CHANG, M. and HOROBITZ,
B.
Fibronectin and fibrinogen structure. J. Biol. Chem., 260, 1811-
1820, 1985
6. MISHIMA,
Y., NAGAO,
F., ISHIBIKI,
K., MATSUDA,
M. and
NAKAMURA,
N. Faktor
XIII
in der Behandlung
postoperatfver
therapierefraktarer
Wundheilungsstorungen.
Chirurg, 55, 803-808,
1984
7. FYE, K.H. and SACK, K.E. Basic and Clinical Immunology, In:
Rheumatic disease, STITES, D.P., STOBO, J.D., FUDENBERG, H.H. and
WELLS, J.V. (eds.), p449, Lange Medical Publications, California,
(1982)
8. HENRIKSSON, P., HENDERNER, U. and NILSSON, I.M. Factor XIII
(fibrin stabilizing factor) in Henoch-Schonlein's purpura. Acta.
Pediatr. Stand., 66, 273-277, 1977
9. HENRIKSSON, P., NILSSON, I.M., OKLSSON, K. and STENBERG, P.
Granulocyte elastase activation and degradation of factor XIII.
Thromb. Res., 18, 343-351, 1980
10. KAMITSUJI, H., TANI, K., YASUI, M., TANIGUCHI, A., TAIRA, K.,
TSUKADA, S., IIDA, Y., KANNKI, H. and FUKUI, H. Activity of blood
coagulation Factor XIII on a prognostic indicator in patients with
Henoch-Schonlein purpura. Eur. J. Pediatr., 146, 519-523, 1987
11. FUKUI, H., KAMITSUJI, H., NAGAO, T., YAMADA, K., AKATSUKA, J.,
INAGAKI, M., SHIKE, S., KOBAYASHI, Y., YOSHIOKA, K., MAKI, S.,
SHIRAHATA, A., MIYAZAKI, S., NAKASHIMA, M. and TANAKA, S. Clinical
evaluation of a pasteurized factor XIII concentrate administration
in Henoch-Schonlein Purpura. Thromb. Res. 56, 667-675, 1989
12. MATSUOKA, M., Hemorrhagic factors and Thrombosis, In: Purpura
Schdnlein-Henoch, ~245-246, Kinbara Shuppan, Tokyo (1981)
13. BOWIE, W.E.J. and OWEN, C.A.Jr Hemostasis and Thrombosis, In:
Non thrombocytopenic vascular disorders, COLMAN, R.W., HIRSH, J,
MARDER, U.J. and SALZMAN, E.W. (eds.), ~816-824,
Lippincott,
Philadelphia, 1987
148
PERMEABILITY INHIBITION BY FXIII
Vol. 71, No. 2
14. ITO, T. and FUJIMAKI,
M. Intergated
handbook
of internal
medicine,
In: SchBnlein-Henoch
purpura,
IMURA, H., OGATA, E.,
TAKAKU, S. and TARUI, S. (eds.), p296-300, Nakayama Shupann, Tokyo
15. WILSON, C.B., COLE, E.H., ZANETTI, M. and MAMPASO, F.M. Basic
and Clinical Immunology, In: Renal disease, STITES, D.P., STOBO,
J-D., FUDENBERG,
H.H. and WELLS, J.V. (eds.), p557-575, Lange
Medical Publication, California (1978)
16. MITSUI,
Y. and
IMAMURA,
J. Isolation
and Culture
for
Functional Cells, In: Endothelial cells., MITSUI, J., TAKAGI, R.,
ICHIHARA, A., SEKIGUTI,
M. and MURAMATSU,
T.(eds.) p227-229,
Maruzen, Tokyo (1987)
17. WICK, G., BAUNDNER, S. and HERZOG, F. Immunofluorescence, p47-
51, Die Medizinishe Verlagsgesellschaft,
Marburg (1987)
18. Yamamoto, T., Chemical Mediators of Inflammation and Immunity,
In; Role of Hageman factor in enhancing vascular permeability.,
CHOEN, S., HAYASHI, H., SAITO, K. and TAKADA, A. (eds.), p129-143,
Academic Press, New York (1986)
19. KATAYAMA, S., SHIONOYA, H. and OHTAKE, S. A new method for
extraction
of extravasated
dye in the skin and influence
of
fasting stress on passive cutaneous anaphylaxis in guinea pigs and
rats. Microbial. Immunol. Biol., 22, 89-101, 1987
20. SHINBO, K., HIRAHARA, K., TAKAHASHI, M. and MATSUISHI,
T.
Suppressive effect of factor XIII on the hemorrhage induced by
anti-endothelial
cell antiserum. Int. J. Hematol., 54(SUPPl 1),
276, 1991
21. KARGES,
H.E. and CLEMENS,
R. Factor XIII: Enzymatic
and
clinical aspects. Behring Inst. Mitt. 82, 43-58, 1988
22. SORIA, A., SORIA, C. and BOULARD, C. Fibrin stabilizing factor
(FXIII) and collagen polymerization. Experientia, 31, 1355-1357,
1975
23. CHOEN, J., BLANKENBERG, T.A., BORDEN, B., KAHN, D.R. and VEIS,
A. Factor XIIIa-catalyzed
crosslinking
of platelet and muscle.
Regulation by nucleotides. Biochem. Biophys. Acta., 628, 365-375,
1980
24. FRANCIS, R.T., MACDONAGH,
J. and MANN, K.G. Factor V is a
substrate for the transamidase factor XIIIa. J. Biol. Chem., 261,
9787-9797, 1986
25. PILGER, E., BERTUCH, H., ULREICH, A. and RAINER, F. Capillary
permeability
in connective
tissue
disease.:
Influence
of
Fibrogammin P-therapy, Thromb. Haemostas. 58, 81, 1987
26, TRYGSTAD, C.W. and STIEHM, E.R. Elevated serum IgA globulin in
anaphylactoid purpura. Pediatrics, 47, 1023-1029, 1971
27. TAKAHASHI,
M., SHINBO, K., HIRAHARA, K. and MATSUISHI,
T.
Effect of activated factor XIII on increase in permeability
of
human umbilical vein endothelial cell layer. The XXIV Congress of
International Society of Hematology in London (abstract), 1992
28. GALOWAY, M.J., MAKIE, M.J. and MACVERRY, B.A. Reduced levels
of factor
XIII in patients
with chronic
inflammatory
bowel
disease. Clin. Lab. Haemat., 5, 427-428, 1983
29. KURATSUJI, T., OKIMA, T., FUKUMOTO, T., SHIMIZU, S., IWASAKI,
Y .,
TOMITA, Y., MEGURO, T. and YAMADA, K. Factor XIII deficiency
in antibiotic-associated
pseudomembranous
colitis
and
its
treatment with Factor XIII concentrate. Haemostas., 11, 229-234,
1982